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University of Florida Thesis Or Dissertation Formatting THE ROLE OF CELL SIGNALING IN POXVIRUS TROPISM: THE CASE OF THE M-T5 HOST-RANGE PROTEIN OF MYXOMA VIRUS By STEVEN JAMES WERDEN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009 1 © 2009 Steven James Werden 2 To my family 3 ACKNOWLEDGMENTS I would like to thank Dr. Grant McFadden for continuous support, encouragement and advice during the course of my PhD. I am truly grateful for all the time and energy he has spent training me to become a better scientist and critical thinker. Without his guidance and persistent help this dissertation would not have been possible. In addition, I am appreciative to my committee members, Drs. Richard Condit, Greg Schultz and Dave Bloom for their encouraging words, fruitful discussion and most importantly, their commitment to helping me succeed. During the past five years, I have had the opportunity and privilege to work with many talented individuals. I would like to acknowledge all members of the McFadden laboratory, both past and present, for creating a work environment that fostered creativity. Special thanks go to Drs.Gen Wang, Steve Nazarian, Marianne Stanford and Fuan Wang for providing technical training. Dr. John Barrett deserves a special mention for his continued mentorship and support. I must not forget to thank those who attended the weekly “Poxaholics” meetings, for providing constructive criticism and a wonderful atmosphere to present data. Thanks go out to Doug Smith for assistance with the confocal microscope and to all who contributed reagents that were used in this dissertation. The University of Florida Medical Guild also deserves recognition for the Research Incentive Award that helped fund my research. I want to thank my family for their support and understanding during the long years of my education. I will never forget my roots and all the wisdom and guidance I received while growing-up. Finally, thank you to my wife; I am forever gratefully for all the support and encouragement she has provided throughout this journey. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...................................................................................................... 4 LIST OF TABLES ................................................................................................................ 9 LIST OF FIGURES ............................................................................................................ 10 LIST OF ABBREVIATIONS .............................................................................................. 12 ABSTRACT........................................................................................................................ 17 CHAPTER 1 INTRODUCTION ........................................................................................................ 20 The Poxviruses ........................................................................................................... 20 Poxvirus Taxonomy ............................................................................................. 20 Characteristics of the Poxviruses ........................................................................ 22 Poxvirus Replication Cycle .................................................................................. 24 Myxoma Virus ............................................................................................................. 26 Historical Relevance of Myxoma Virus ................................................................ 27 Pathogenesis of Myxoma Virus ........................................................................... 28 Myxomatosis Prevention, Control and Vaccination ............................................ 29 Poxvirus Immunoregulatory Genes Dictate Tropism ................................................. 30 Orthopoxvirus Host-Range Genes ...................................................................... 33 Myxoma Virus Host-Range Genes ...................................................................... 34 M-T5 is a Host-Range Gene Encoded by Myxoma Virus ......................................... 35 Myxoma Virus Replication in Rabbit Lymphocytes is Dependent Upon M-T5 .. 36 M-T5 Inhibits the Induction of Apoptosis in Rabbit Lymphocytes ...................... 37 M-T5 is a Potent Virulence Factor in European Rabbits .................................... 38 Ankyrin-Repeat Protein Superfamily .......................................................................... 39 Poxvirus Encoded Ankyrin-Repeat Proteins ....................................................... 40 Most ANK-repeat Poxviral Proteins Contain a Conserved F-Box Domain ........ 41 F-box Proteins are Specific Factors of SCF Ligases .......................................... 41 M-T5 Binds to Cullin 1.......................................................................................... 42 Expanding Myxoma Virus Tropism ............................................................................ 43 Myxoma Virus as a Candidate for Oncolytic Virotherapy ................................... 45 Intracellular Signaling Manipulation: The Role of M-T5 ...................................... 46 Dissertation Objectives ............................................................................................... 49 2 MATERIALS AND METHODS ................................................................................... 50 DNA Methods .............................................................................................................. 50 Reagents .............................................................................................................. 50 Polymerase Chain Reaction ................................................................................ 50 5 Purification of PCR Products ............................................................................... 51 Restriction Enzyme Digestion of DNA ................................................................. 51 DNA Ligation ........................................................................................................ 51 Transformation of E. coli ...................................................................................... 52 Plasmid Production and Purification.................................................................... 52 DNA Sequencing.................................................................................................. 53 Plasmids ............................................................................................................... 53 Cell Culture ................................................................................................................. 55 Reagents .............................................................................................................. 55 Cell Culture ........................................................................................................... 56 Viruses and Infection ........................................................................................... 56 Transfection of Mammalian Cells ........................................................................ 58 siRNA Transfections ............................................................................................ 58 Drug Inhibition Studies ......................................................................................... 58 Protein Methods .......................................................................................................... 59 Reagents .............................................................................................................. 59 Cell Lysis .............................................................................................................. 60 Immunoprecipitation Assay.................................................................................. 61 GST Pull-Down Assay ......................................................................................... 61 SDS-PAGE ........................................................................................................... 61 Immunoblot Analysis ............................................................................................ 62 Yeast Two-Hybrid Screening ............................................................................... 62 AlphaScreen Binding Protocol ............................................................................. 62 Nuclear Extraction ................................................................................................ 63 Microscopy - Cellular Localization.............................................................................. 64 Reagents .............................................................................................................. 64 Fixation, Permeabilization and Blocking ............................................................. 64 Immunostaining .................................................................................................... 64 3 M-T5 IS A NOVEL ADAPTOR THAT COORDINATELY LINKS THE CELLULAR SIGNALING PATHWAYS MEDIATED BY AKT AND SKP1 IN VIRUS- INFECTED CELLS ..................................................................................................... 66 Introduction ................................................................................................................. 66 Results .......................................................................................................................
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